Inhibition of endothelial nitric oxide synthase by the lipid phosphatase PTEN

Jarrod E. Church, Jin Qian, Sanjiv Kumar, Stephen M. Black, Richard C. Venema, Andreas Papapetropoulos, David J R Fulton

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a lipid phosphatase that functions as a negative regulator of the phosphoinositide-3-kinase (PI3K) pathway. The present study sought to examine in depth the interaction between PTEN and eNOS activity. Co-expression of eNOS and PTEN in COS-7 cells significantly decreased NO production compared to eNOS alone, while co-expression of eNOS and the dominant negative mutant PTEN(C124A) significantly increased NO production. Upon examination of the putative eNOS phosphorylation sites, phosphorylation of S116, T497, S617, S635 and S1179 was decreased by PTEN co-expression, while the dominant negative PTEN(C124A) produced an increase in phosphorylation of all sites except S116 and S635. A myristoylation-deficient eNOS construct with little dependence on phosphorylation state (G2AeNOS) was not significantly affected by co-expression with either PTEN or PTEN(C124A). Likewise, an eNOS construct with a triple phospho-null mutation (S617A, S635A and S1179A) was also unaffected by co-expression with either PTEN or PTEN(C124A). Purified PTEN or PTEN(C124A) failed to interact with purified eNOS in vitro, arguing against a direct interaction between PTEN and eNOS. When the PTEN constructs were expressed in human aortic endothelial cells (HAECs), PTEN significantly decreased NO production and PTEN(C124A) increased it, and both S617 and S1179 were altered by co-expression with the PTEN constructs. Increased expression of PTEN in endothelial cells did not influence superoxide production. We conclude that PTEN is a regulator of eNOS function both when expressed in COS-7 cells and in human endothelial cells, and does so via its effects on the PI3K/Akt pathway.

Original languageEnglish (US)
Pages (from-to)191-198
Number of pages8
JournalVascular Pharmacology
Volume52
Issue number5-6
DOIs
StatePublished - May 1 2010
Externally publishedYes

Fingerprint

PTEN Phosphohydrolase
Nitric Oxide Synthase Type III
Phosphorylation
Lipids
1-Phosphatidylinositol 4-Kinase
Endothelial Cells
COS Cells
Chromosomes, Human, Pair 10
Phosphoric Monoester Hydrolases
Superoxides
Mutation

Keywords

  • Akt
  • eNOS
  • Nitric oxide
  • PTEN
  • Vascular

ASJC Scopus subject areas

  • Pharmacology
  • Molecular Medicine
  • Physiology

Cite this

Church, J. E., Qian, J., Kumar, S., Black, S. M., Venema, R. C., Papapetropoulos, A., & Fulton, D. J. R. (2010). Inhibition of endothelial nitric oxide synthase by the lipid phosphatase PTEN. Vascular Pharmacology, 52(5-6), 191-198. https://doi.org/10.1016/j.vph.2009.11.007

Inhibition of endothelial nitric oxide synthase by the lipid phosphatase PTEN. / Church, Jarrod E.; Qian, Jin; Kumar, Sanjiv; Black, Stephen M.; Venema, Richard C.; Papapetropoulos, Andreas; Fulton, David J R.

In: Vascular Pharmacology, Vol. 52, No. 5-6, 01.05.2010, p. 191-198.

Research output: Contribution to journalArticle

Church, JE, Qian, J, Kumar, S, Black, SM, Venema, RC, Papapetropoulos, A & Fulton, DJR 2010, 'Inhibition of endothelial nitric oxide synthase by the lipid phosphatase PTEN', Vascular Pharmacology, vol. 52, no. 5-6, pp. 191-198. https://doi.org/10.1016/j.vph.2009.11.007
Church, Jarrod E. ; Qian, Jin ; Kumar, Sanjiv ; Black, Stephen M. ; Venema, Richard C. ; Papapetropoulos, Andreas ; Fulton, David J R. / Inhibition of endothelial nitric oxide synthase by the lipid phosphatase PTEN. In: Vascular Pharmacology. 2010 ; Vol. 52, No. 5-6. pp. 191-198.
@article{2fe211b5882548c98a1a5521a3312b03,
title = "Inhibition of endothelial nitric oxide synthase by the lipid phosphatase PTEN",
abstract = "PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a lipid phosphatase that functions as a negative regulator of the phosphoinositide-3-kinase (PI3K) pathway. The present study sought to examine in depth the interaction between PTEN and eNOS activity. Co-expression of eNOS and PTEN in COS-7 cells significantly decreased NO production compared to eNOS alone, while co-expression of eNOS and the dominant negative mutant PTEN(C124A) significantly increased NO production. Upon examination of the putative eNOS phosphorylation sites, phosphorylation of S116, T497, S617, S635 and S1179 was decreased by PTEN co-expression, while the dominant negative PTEN(C124A) produced an increase in phosphorylation of all sites except S116 and S635. A myristoylation-deficient eNOS construct with little dependence on phosphorylation state (G2AeNOS) was not significantly affected by co-expression with either PTEN or PTEN(C124A). Likewise, an eNOS construct with a triple phospho-null mutation (S617A, S635A and S1179A) was also unaffected by co-expression with either PTEN or PTEN(C124A). Purified PTEN or PTEN(C124A) failed to interact with purified eNOS in vitro, arguing against a direct interaction between PTEN and eNOS. When the PTEN constructs were expressed in human aortic endothelial cells (HAECs), PTEN significantly decreased NO production and PTEN(C124A) increased it, and both S617 and S1179 were altered by co-expression with the PTEN constructs. Increased expression of PTEN in endothelial cells did not influence superoxide production. We conclude that PTEN is a regulator of eNOS function both when expressed in COS-7 cells and in human endothelial cells, and does so via its effects on the PI3K/Akt pathway.",
keywords = "Akt, eNOS, Nitric oxide, PTEN, Vascular",
author = "Church, {Jarrod E.} and Jin Qian and Sanjiv Kumar and Black, {Stephen M.} and Venema, {Richard C.} and Andreas Papapetropoulos and Fulton, {David J R}",
year = "2010",
month = "5",
day = "1",
doi = "10.1016/j.vph.2009.11.007",
language = "English (US)",
volume = "52",
pages = "191--198",
journal = "Vascular Pharmacology",
issn = "1537-1891",
publisher = "Elsevier Inc.",
number = "5-6",

}

TY - JOUR

T1 - Inhibition of endothelial nitric oxide synthase by the lipid phosphatase PTEN

AU - Church, Jarrod E.

AU - Qian, Jin

AU - Kumar, Sanjiv

AU - Black, Stephen M.

AU - Venema, Richard C.

AU - Papapetropoulos, Andreas

AU - Fulton, David J R

PY - 2010/5/1

Y1 - 2010/5/1

N2 - PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a lipid phosphatase that functions as a negative regulator of the phosphoinositide-3-kinase (PI3K) pathway. The present study sought to examine in depth the interaction between PTEN and eNOS activity. Co-expression of eNOS and PTEN in COS-7 cells significantly decreased NO production compared to eNOS alone, while co-expression of eNOS and the dominant negative mutant PTEN(C124A) significantly increased NO production. Upon examination of the putative eNOS phosphorylation sites, phosphorylation of S116, T497, S617, S635 and S1179 was decreased by PTEN co-expression, while the dominant negative PTEN(C124A) produced an increase in phosphorylation of all sites except S116 and S635. A myristoylation-deficient eNOS construct with little dependence on phosphorylation state (G2AeNOS) was not significantly affected by co-expression with either PTEN or PTEN(C124A). Likewise, an eNOS construct with a triple phospho-null mutation (S617A, S635A and S1179A) was also unaffected by co-expression with either PTEN or PTEN(C124A). Purified PTEN or PTEN(C124A) failed to interact with purified eNOS in vitro, arguing against a direct interaction between PTEN and eNOS. When the PTEN constructs were expressed in human aortic endothelial cells (HAECs), PTEN significantly decreased NO production and PTEN(C124A) increased it, and both S617 and S1179 were altered by co-expression with the PTEN constructs. Increased expression of PTEN in endothelial cells did not influence superoxide production. We conclude that PTEN is a regulator of eNOS function both when expressed in COS-7 cells and in human endothelial cells, and does so via its effects on the PI3K/Akt pathway.

AB - PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a lipid phosphatase that functions as a negative regulator of the phosphoinositide-3-kinase (PI3K) pathway. The present study sought to examine in depth the interaction between PTEN and eNOS activity. Co-expression of eNOS and PTEN in COS-7 cells significantly decreased NO production compared to eNOS alone, while co-expression of eNOS and the dominant negative mutant PTEN(C124A) significantly increased NO production. Upon examination of the putative eNOS phosphorylation sites, phosphorylation of S116, T497, S617, S635 and S1179 was decreased by PTEN co-expression, while the dominant negative PTEN(C124A) produced an increase in phosphorylation of all sites except S116 and S635. A myristoylation-deficient eNOS construct with little dependence on phosphorylation state (G2AeNOS) was not significantly affected by co-expression with either PTEN or PTEN(C124A). Likewise, an eNOS construct with a triple phospho-null mutation (S617A, S635A and S1179A) was also unaffected by co-expression with either PTEN or PTEN(C124A). Purified PTEN or PTEN(C124A) failed to interact with purified eNOS in vitro, arguing against a direct interaction between PTEN and eNOS. When the PTEN constructs were expressed in human aortic endothelial cells (HAECs), PTEN significantly decreased NO production and PTEN(C124A) increased it, and both S617 and S1179 were altered by co-expression with the PTEN constructs. Increased expression of PTEN in endothelial cells did not influence superoxide production. We conclude that PTEN is a regulator of eNOS function both when expressed in COS-7 cells and in human endothelial cells, and does so via its effects on the PI3K/Akt pathway.

KW - Akt

KW - eNOS

KW - Nitric oxide

KW - PTEN

KW - Vascular

UR - http://www.scopus.com/inward/record.url?scp=77951022204&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77951022204&partnerID=8YFLogxK

U2 - 10.1016/j.vph.2009.11.007

DO - 10.1016/j.vph.2009.11.007

M3 - Article

C2 - 19962452

AN - SCOPUS:77951022204

VL - 52

SP - 191

EP - 198

JO - Vascular Pharmacology

JF - Vascular Pharmacology

SN - 1537-1891

IS - 5-6

ER -